Identification and characterization of glyceraldehyde 3-phosphate dehydrogenase from Fasciola gigantica

Abstract

Fasciola gigantica is an important food-borne trematode responsible for the hepatobiliary disease, commonly known as fascioliasis. In F. gigantica, the glyceraldehyde 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the glycolytic pathway and catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD+ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the sister fluke Fasciola hepatica GAPDH. The gapdh gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~ 37-kDa subunit under non-dissociating conditions at 300 mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD+ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD+) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in the case of FgGAPDH, similar to the human GAPDH. Thus, the conformation of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.

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Abbreviations

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

FgGAPDH:

F. gigantica glyceraldehyde 3-phosphate dehydrogenase

SEC:

Size exclusion chromatography

G-3-P:

d-glyceraldehyde-3-phosphate

DTT:

Dithiothreitol

HEPES:

(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)

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PBC and RS carried out all the experiments. PBC, RS, and TT analyzed the data, conceived the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.

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Correspondence to Timir Tripathi.

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Chetri, P.B., Shukla, R. & Tripathi, T. Identification and characterization of glyceraldehyde 3-phosphate dehydrogenase from Fasciola gigantica. Parasitol Res 118, 861–872 (2019). https://doi.org/10.1007/s00436-019-06225-w

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Keywords

  • Fasciola gigantica
  • Liver fluke
  • Glyceraldehyde 3-phosphate dehydrogenase
  • Activity
  • Modeling
  • Docking
  • Quenching